Overview Over the past decade, systematic studies from our laboratory using adolescent rhesus macaques have shown that social stress increases alcohol consumption and that during nonstressful periods, interindividual rates of alcohol consumption are positively correlated with interindividual trait-like anxiety and hypothalamic-pituitary-adrenal (HPA) axis output. HPA Axis and Alcohol To date, studies from our laboratory have shown that the strongest predictor of excessive alcohol intake is the response of the HPA Axis, with high levels of cortisol being highly predictive of alcohol intake. Chronic activation of the neuroendocrine stress axis via high alcohol intake contributes to allostatic changes at the level of the brain, which ultimately can lead to alcoholism. In studies reported last year, genetic and early experience, as well as gender, were shown to affect the HPA Axis and, ultimately, voluntary alcohol intake or response to a uniform 2 gm/kg dose of alcohol. Corticotropin-releasing hormone (CRH) is the primary neuropeptide responsible for activation of the HPA axis. As such, the CRH gene is a good candidate for investigating genetic variation as it relates to vulnerability to anxiety and mood disorders, neuroendocrine stress axis dysregulation, and alcoholism. Dr. Christina Barr, along with a graduate student, Rachel Dvoskin, have completed sequencing the CRH gene and promoter region in rhesus and have screened both the rhesus and human CRH genes for sequence variants, identifying a number of polymorphisms. They have found that the rhesus and human CRH genes share 97% identity in coding regions and 94% identity in noncoding regions, suggesting high homology. There are a number of single nucleotide polymorphisms (SNPs) found in both the rhesus and the human CRH gene promoters. Among the variants that Dr. Barr and Ms. Dvoskin discovered in rhesus macaques is a SNP (-248C>T) that is present within a region that is critical to regulation of CRH gene transcription. Dr. Barr performed cross-species analysis of the 400 bp region surrounding this variant using sequences obtained either in our laboratory or from various public databases. In so doing, she demonstrated almost 100% sequence identity among rat, mouse, dog, sheep, rhesus and human, suggesting this region to be under selective pressure. In rhesus, the ?248 T polymorphism creates a TATA-like element, and Dr. Barr found the ?248T allele to be associated with increased ACTH and cortisol responses to social separation stress in infant macaques. She also found there to be an interactive effect with early rearing in that ACTH and cortisol levels are especially high among carriers of the -248T allele that exposed to early adversity in the form of parental deprivation. Using electrophoretic mobility shift assays, Dr. Barr demonstrated that another rhCRH promoter variant (-2232 C>G) disrupts one of the glucocorticoid response elements (GREs), which are known to be critical to feedback control of CRH expression by corticosteroids. This variant is in allelic identity with a number of other polymorphisms spanning the rhCRH gene, producing a specific haplotype cluster (rhCRH-A2) that is present in 15% of the animals in our colony. By performing in vitro studies using a pDsRed expression system in COS-7 cells, Dr. Barr and Ms. Dvoskin demonstrated higher basal and stimulated promoter activity in the rhCRH-A2 construct compared to the rhCRH-A1 (nonvariant) construct. Unlike the CRH?248T polymorphism, the rhCRH-A2 haplotype is associated with increased basal levels of ACTH, with no observable differences during social separation stress, suggesting differential regulation of basal and stress-modulated ACTH output. The rhCRH-A2 haplotype is also associated with increased HPA response to alcohol, and animals carrying the rhCRH-A2 haplotype exhibit increased alcohol consumption with consecutive exposures, consistent with the putative role of CRH and the HPA axis in promoting escalation of alcohol and drug intake. Maternal Behavior and Physiology To understand how situational and temperament differences in behavior might influence the stress system, Dr. Schwandt began a series of analyses investigating maternal behavior and stress. In her role as our Data Manager and as an independent researcher, Dr. Schwandt found that maternal experience (parity) mediates the relationship between physiology and behavior. In month 1, the early postpartum period (during which the mother is adapting to the stress of a new infant), affiliative social contact between mothers and other group members was positively correlated with ACTH levels in primiparous mothers and positively correlated with HVA levels in multiparous mothers. These results suggest that mothers may be seeking social support during a relatively stressful period, and that among first time mothers such social support is insufficient to ameliorate stress-elevated HPA levels. Another possibility may have been that other group members were seeking out new mothers to obtain access to the infant, and this may have caused stress in some first-time mothers, who tend to be more overly protective of their infants than experienced mothers. Studies of first time mothers in the natural setting have shown that it is not unusual for inexperienced mothers to lose access to their infant once another female picks it up. For multiparous mothers, on the other hand, such interactions may be positive and supportive, since they can readily get their infant back. In month 5, the beginning period of weaning, which is a potentially stressful time for both mother and infant, rejection of the infant was positively correlated with ACTH levels in primiparous but not multiparous mothers. Studies show that when compared to the multiparous mothers, primiparous mothers have difficulty in the weaning transition, potentially resulting in more stress in the mother-infant relationship and consequent high HPA activity. Other Measures of Stress Rhesus infants typically respond to periods of social separation by producing frequent vocalizations. In a study by Dr. Becker and our NICHD colleague John Newman, we examined the relationship between cortisol and vocal activity during brief periods of separation from the infants? attachment source (mother or surrogate mother) over the first 4 weeks of life. Multiple regression analyses revealed a positive relationship between cortisol levels and vocal behavior, but only at day 30. This relationship was stronger in the nursery-reared (r= 0.80) when compared to the mother-reared (r= 0.40) infants. These results suggest that adrenal activity and vocal behavior resulting from brief social separations depends on developmental age and rearing experience.